Effect of cajuputs candy formulas on competitive capacity of streptococcus sanguinis upon streptococcus mutans in multispecies biofilm.

Abstract

Cajuputs candy development as a functional food has been shifted to maintain the oral health. Cajuputs candy is classified as hard candy which utilized cajuput and peppermint oil as its flavor components. Streptococcus sanguinis is a health-associated species which can control the growth of cariogenic species, S. mutans. These two oral Streptococci have antagonistic interaction which plays a role in preventing dental caries formation. The aim of this research is to determine the effects of two type of cajuputs candy, i.e. Sucrose Cajuputs Candy (SCC) and Non-Sucrose Cajuputs Candy (NSCC), on competitive capacity of S. sanguinis upon S. mutans in multispecies (S. sanguinis+S. mutans) biofilm, which is indicated by its ability to form biofilm, control the growth of S. mutans, and expressed spxB mRNA. The spxB mRNA expression reflected hydrogen peroxide (H2O2) production by S. sanguinis. Volatile constituents of cajuputs candy’s flavors were analyzed using Gas Chromatography-Mass Spectrometry (GC-MS). In vitro biofilm assay with optical density (OD) measurement was used to examine the potency of cajuputs candy in inhibiting the formation of multispecies biofilm. Quantitative real-time polymerase chain reaction (real-time PCR) techniques were applied for quantification of spxB mRNA and relative amount of total DNA bacterium in the biofilms. Several reported antimicrobial components, i.e. 1,8-cineole (23,67%), α-terpineol (9,17%), menthol (7,29%), β-caryophyllene (6,78%), menthone (3,30%), and terpinen-4-ol (1,86%) were detected in SCC. Meanwhile, the reported antimicrobial components, i.e. menthol (13,32%), α-terpineol (9,97%), 1,8-cineole (8,48%), β-caryophyllene (7,64%), menthone (4,43%) and terpinen-4-ol (1,28%) were also detected in NSCC. The biofilm assay showed that the exposures of SSC and NSCC were effective in inhibiting multispecies biofilm. Biofilm inhibition caused by SCC was 59,2% and NSCC was 68,2%. Moreover, SCC and NSCC exposures also decreased the relative amount of total DNA in multispecies biofilm to be 1,20% and 1,04% compared to the control (100%), respectively. These reductions were indicating that SCC and NSCC could inhibit biofilm formation of S. sanguinis and S. mutans, in vitro. Exposures of candy formula without flavor, i.e sucrose candy without flavor and non-sucrose candy without flavor, also affected biofilm formation. The ability to form biofilm affected by sucrose candy and non-sucrose candy without flavor exposures were decreased 36,9% and 47,1% compared with the control, respectively. However, sucrose candy and non-sucrose candy without flavor exposures were induced bacterial growth with percentage of total DNA in the biofilm were 184,77% and 221,29% increase compared with the control, respectively. It seems that utilization of sucrose without addition of antibacterial component, e.g. essential oil as flavoring agent, could induce bacterial growth. Even a non-sucrose utilization, in this study is isomalt - a non-cariogenic polyol-, with a long exposure time (approx. 20 hours) in this research could be fermented by these Streptococci and can be used for their growth. This indicated that cajuput and peppermint oil in SCC and NSCC showed antibacterial activity. The proportion of S. sanguinis DNA in multispecies biofilms was lower than S. mutans DNA in all samples, which ranged from 35 to 42% for S. sanguinis and 58 to 65% for S. mutans. This bacterial DNA proportion was not statistically significant compared with the control, suggest that cajuputs candy exposures did not affect S. sanguinis and S. mutans interaction. Analysis of spxB mRNA of S. sanguinis shows that SCC and NSCC exposures could maintain spxB mRNA expression level relatively at 0.5 fold and 0.9 fold compared with the control (1,0 fold), respectively. The expression of spxB mRNA was decreased to be 0.3 fold after sucrose candy without flavor exposure, while exposures of non-sucrose candy without flavor increase spxB mRNA expression to be 1.1 fold. The detected spxB mRNA in the biofilms indicated that cajuputs candy formulas exposures did not affect S. sanguinis ability to produce H2O2 and could be considered as competitive against S. mutans. Both cajuputs candies (SCC and NSCC) have the potency in maintaining the competitive capacity of S. sanguinis upon S. mutans in multispecies biofilm.